RNAseq of rat feces Day 21
DNA was extracted from the rat feces and cecum using DNeasy PowerLyzer PowerSoil kit according to manufacturer protocol, and DNA concentration measured by Qbit dsDNA HF kit. In the extracted samples the v3 region of the 16S RNA gene was amplified in one PCR reactions including fusion primers with the Ion Torrent sequencing adaptors and barcode. (PBU 5-A-adapter-TCAG-barcode-CCTACGGGAGGCAGCAG-3) and reverse primer (PBR 5-trP1-adapter-ATTACCGCGGCTGCTGG-3) in 20 l total reaction volume. PCR products cleaned using HighPrep PCR magnetic beads and cleaned PCR products again measured for DNA concentration using Qubit dsDNA HS assay. For sequencing equimolar amount of each PCR product was mixed and 16S rRNA gene sequencing was performed on an IONS5 system.